Note: Descriptions are shown in the official language in which they were submitted.
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COMBINATION
FIELD OF THE INVENTION
The present invention relates to a method of treating cancer in a mammal and
to
combinations useful in such treatment. In particular, the method relates to a
novel
combination comprising the MEK inhibitor: N-{3-[3-cyclopropyl-5-[(2-fluoro-4-
iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-
d]pyrimidin-
1(2H)-yl]phenyl}acetamide, or a pharmaceutically acceptable salt or solvate
thereof, and
the P13K inhibitor: 2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-
quinolinyl]-3-
pyridinyl}benzenesulfonamide, or a pharmaceutically acceptable salt thereof,
pharmaceutical compositions comprising the same, and methods of using such
combinations in the treatment of cancer.
BACKGROUND OF THE INVENTION
Effective treatment of hyperproliferative disorders including cancer is a
continuing
goal in the oncology field. Generally, cancer results from the deregulation of
the normal
processes that control cell division, differentiation and apoptotic cell
death. Apoptosis
(programmed cell death) plays essential roles in embryonic development and
pathogenesis of various diseases, such as degenerative neuronal diseases,
cardiovascular diseases and cancer. One of the most commonly studied pathways,
which
involves kinase regulation of apoptosis, is cellular signaling from growth
factor receptors
at the cell surface to the nucleus (Crews and Erikson, Cell, 74:215-17, 1993).
An important large family of enzymes is the protein kinase enzyme family.
Currently, there are about 500 different known protein kinases. Protein
kinases serve to
catalyze the phosphorylation of an amino acid side chain in various proteins
by the
transfer of the y-phosphate of the ATP-Mg2+ complex to said amino acid side
chain.
These enzymes control the majority of the signaling processes inside cells,
thereby
governing cell function, growth, differentiation and destruction (apoptosis)
through
reversible phosphorylation of the hydroxyl groups of serine, threonine and
tyrosine
residues in proteins. Studies have shown that protein kinases are key
regulators of many
cell functions, including signal transduction, transcriptional regulation,
cell motility, and
cell division. Several oncogenes have also been shown to encode protein
kinases,
suggesting that kinases play a role in oncogenesis. These processes are highly
regulated, often by complex intermeshed pathways where each kinase will itself
be
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regulated by one or more kinases. Consequently, aberrant or inappropriate
protein kinase
activity can contribute to the rise of disease states associated with such
aberrant kinase
activity including benign and malignant proliferative disorders as well as
diseases
resulting from inappropriate activation of the immune and nervous systems. Due
to their
physiological relevance, variety and ubiquitousness, protein kinases have
become one of
the most important and widely studied family of enzymes in biochemical and
medical
research.
The protein kinase family of enzymes is typically classified into two main
subfamilies: Protein Tyrosine Kinases and Protein Serine/Threonine Kinases,
based on
the amino acid residue they phosphorylate. The protein serine/threonine
kinases (PSTK),
includes cyclic AMP- and cyclic GMP-dependent protein kinases, calcium and
phospholipid dependent protein kinase, calcium- and calmodulin-dependent
protein
kinases, casein kinases, cell division cycle protein kinases and others. These
kinases are
usually cytoplasmic or associated with the particulate fractions of cells,
possibly by
anchoring proteins. Aberrant protein serine/threonine kinase activity has been
implicated
or is suspected in a number of pathologies such as rheumatoid arthritis,
psoriasis, septic
shock, bone loss, many cancers and other proliferative diseases. Accordingly,
serine/threonine kinases and the signal transduction pathways which they are
part of are
important targets for drug design. The tyrosine kinases phosphorylate tyrosine
residues.
Tyrosine kinases play an equally important role in cell regulation. These
kinases include
several receptors for molecules such as growth factors and hormones, including
epidermal growth factor receptor, insulin receptor, platelet derived growth
factor receptor
and others. Studies have indicated that many tyrosine kinases are
transmembrane
proteins with their receptor domains located on the outside of the cell and
their kinase
domains on the inside. Much work is also in progress to identify modulators of
tyrosine
kinases as well.
Mitogen-activated protein kinase (MAPK) Kinase/extracellular signal-regulated
kinase (ERK) kinase (hereinafter referred to as MEK) is known to be involved
in the
regulation of numerous cellular processes. The Raf family (B-Raf, C-Raf etc.)
activates
the MEK family (MEK-1, MEK-2 etc.) and the MEK family activates the ERK family
(ERK-
1 and ERK-2). Broadly, the signaling activity of the RAF/MEK/ERK pathway
controls
mRNA translation. This includes genes related to the cell cycle. Hence,
hyperactivation
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of this pathway can lead to uncontrolled cell proliferation. Deregulation of
the
RAF/MEK/ERK pathway by ERK hyperactivation is seen in approximately 30% of all
human malignancies (Allen, LF, et al. Semin. Oncol. 2003. 30(5 Suppl 16):105-
16).
RAS, which can signal through both the P13K/AKT and RAF/MEK/ERK, has a mutated
oncogenic protein in 15% of all cancers (Davies, H. et al. Nature. 2002.
417:949-54).
Also, activating BRAF mutations have been identified at a high frequency in
specific
tumor types (e.g., melanomas) (Davies, H. et al. Nature. 2002. 417:949-54).
Although
activating mutations in MEK itself don't appear to frequently occur in human
cancers,
MEK is thought to be an important drug target for treating human cancer
because of its
central role in the ERK pathway. Further, MEK inhibitory activity effectively
induces
inhibition of ERK1/2 activity and suppression of cell proliferation (The
Journal of Biological
Chemistry, vol. 276, No. 4, pp. 2686-2692, 2001), and the compound is expected
to show
effects on diseases caused by undesirable cell proliferation, such as tumor
genesis
and/or cancer.
The phosphoinositide 3-kinase (P13K) pathway is among the most commonly
activated pathways in human cancer. The function and importance of this
pathway in
tumorigenesis and tumor progression is well established (Samuels & Ericson.
Curr. Opp
in Oncology, 2006. 18: 77-82). P13K-AKT signaling appears to be a pivotal
modulator of
cell survival, proliferation and metabolism. This includes the activation of
mammalian
target of rapamycin (mTOR), a P13K protein family member and direct regulator
of cell
growth and translation. Thus, the deregulation of P13K/AKT/mTOR signaling in
tumors
contributes to a cellular phenotype that demonstrates numerous hallmarks of
malignancies, which includes unlimited reproductive potential and the evasion
of
apoptosis (Hanahan & Weinberg, Cell. 2000. 100:57-70).
The P13K family consists of 15 proteins that share sequence homology,
particularly within their kinase domains; however; they have distinct
substrate specificities
and modes of regulation (Vivanco & Sawyers. Nat. Rev. Cancer, 2002.2:489-501).
Class
1 P13-kinases phosphorylate inositol-containing lipids, known as
phosphatidylinositols
(Ptdlns) at the 3 position. The primary substrate of Class I family members,
Ptdlns-4, 5-
P2 (PIP2) is converted to Ptdlns-3, 4, 5-P3 (PIP3) by these kinases. PIP3 is a
critical
second messenger which recruits proteins that contain pleckstrin homology
domains to
the cell membrane where they are activated. The most studied of these proteins
is AKT
which promotes cell survival, growth, and proliferation. Upon activation, AKT
moves to
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the cytoplasm and nucleus where it phosphorylates numerous substrates,
including
mTOR (TORC1). In addition to AKT, P13K activates other pathways that are
implicated in
carcinogenesis such as PDK1, CDC42 and RAC1 (Samuels & Ericson. Curr. Opp in
Oncology, 2006. 18: 77-82).
In the study of human tumors, activation of the P13K/AKT/mTOR signaling
pathway can occur via numerous mechanisms. Genetic deregulation of the pathway
is
common and can occur in a number of ways (reviewed in Samuels & Ericson. Curr.
Opp
in Oncology, 2006. 18: 77-82). Activating mutations of the PIK3CA gene (coding
for the
p110a catalytic subunit of P13K) occur in a significant percentage of human
tumors
including breast, ovarian, endometrial, and colorectal cancer. Activating DNA
amplifications of this gene also occur less frequently in a number of
different tumor types.
Mutations in the p85a regulatory subunit of P13K (PIK3R1), which are thought
to disrupt
the C2-iSH2 interaction between PIK3R1 and PIK3CA, occur in ovarian,
glioblastoma and
colorectal cancer. The tumor suppressor PTEN, which dephosphorylates PIP3 to
generate PIP2 and thus acts as an inhibitor of the P13K pathway, is commonly
mutated,
deleted, or epigenetically silenced. Finally, the pathway can also be
genetically activated
downstream of P13K by DNA amplification or mutation of AKT; however these
genetic
events occur much less frequently in human cancer. Inhibiting P13K isoforms,
particularly
P13Ka, are known to be useful in the treatment of cancer (see for example WO
05/121142, WO 08/144463, WO 08/144464, WO 07/136940).
SUMMARY OF THE INVENTION
One embodiment of this invention provides a combination comprising:
(i) a compound of Structure (1):
F '
~ I
O HN
N NiCH3
O N O
CH3
H3C N
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(I)
N-{3-[3-cyclopropyl-5-[(2-fl uoro-4-iodophenyl)amino]-6,8-d imethyl-2,4,7-
trioxo-3,4, 6, 7-
tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl]phenyl}acetamide (hereinafter
Compound A)
or a pharmaceutically acceptable salt thereof; and
(ii) a compound of Structure (11):
F
F
- /N \N
O-S=0
-O
N
N
(11)
2,4-d ifluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-qu inoIinyl]-3-
pyridinyl}benzenesulfonamide (hereinafter Compound B)
or a pharmaceutically acceptable salt thereof.
One embodiment of this invention provides a method of treating cancer in a
human in need thereof which comprises the in vivo administration of a
therapeutically
effective amount of a combination of Compound A, or a pharmaceutically
acceptable salt
or solvate, suitably the dimethyl sulfoxide solvate, thereof, and Compound B,
or a
pharmaceutically acceptable salt thereof, to such human.
One embodiment of this invention provides a method of treating cancer in a
human in need thereof which comprises the in vivo administration of a
therapeutically
effective amount of a combination of Compound A, or a pharmaceutically
acceptable salt
or solvate, suitably the dimethyl sulfoxide solvate, thereof, and Compound B,
or a
pharmaceutically acceptable salt thereof, to such human,
wherein the combination is administered within a specified period, and
wherein the combination is administered for a duration of time.
One embodiment of this invention provides a method of treating cancer in a
human in need thereof which comprises the in vivo administration of a
therapeutically
effective amount of a combination of Compound A, or a pharmaceutically
acceptable salt
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or solvate, suitably the dimethyl sulfoxide solvate, thereof, and Compound B,
or a
pharmaceutically acceptable salt thereof, to such human,
Wherein compounds A and B are administered sequentially.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to combinations that exhibit anti proliferative
activity.
Suitably, the method relates to methods of treating cancer by the co-
administration of N-
{3-[3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-
3,4,6,7-
tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl]phenyl}acetamide (Compound A), or a
pharmaceutically acceptable salt or solvate, suitably the dimethyl sulfoxide
solvate
thereof, which compound is represented by Structure I:
F '
~ I
O HN
CH
611,
s
N NN
O)" N O
O CHs
~
H3CA N \
H
(I)
and 2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-
pyridinyl}benzenesulfonamide (Compound B), or a pharmaceutically acceptable
salt
thereof; which compound is represented by the following structure
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F
F
O=S=O
NN
-O
N
N
(II)
Compound A, also known as N-{3-[3-cyclopropyl-5-(2-fluoro-4-iodo-phenylamino)-
6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-
yl]phenyl}acetamide is disclosed and claimed, along with pharmaceutically
acceptable
salts and solvates thereof, as being useful as an inhibitor of MEK activity,
particularly in
treatment of cancer, in International Application No. PCT/JP2005/011082,
having an
International filing date of June 10, 2005; International Publication Number
WO
2005/121142 and an International Publication date of December 22, 2005, the
entire
disclosure of which is hereby incorporated by reference, Compound A is the
compound of
Example 4-1. Compound A can be prepared as described in International
Application No.
PCT/JP2005/011082. Compound A can be prepared as described in United States
Patent Publication No. US 2006/0014768, Published January 19, 2006, the entire
disclosure of which is hereby incorporated by reference.
Suitably, Compound A is in the form of a dimethyl sulfoxide solvate. Suitably,
Compound A is in the form of a sodium salt. Suitably, Compound A is in the
form of a
solvate selected from: hydrate, acetic acid, ethanol, nitromethane,
chlorobenzene, 1-
pentanol, isopropyl alcohol, ethylene glycol and 3-methyl-1-butanol. These
solvates and
salt forms can be prepared by one of skill in the art from the description in
International
Application No. PCT/JP2005/011082 or United States Patent Publication No. US
2006/0014768.
Compound B is disclosed and claimed, along with pharmaceutically acceptable
salts thereof, as being useful as an inhibitor of P13K activity, particularly
in treatment of
cancer, in International Application No. PCT/US2008/063819, having an
International
filing date of May 16, 2008; International Publication Number WO 2008/144463
and an
International Publication date of November 27, 2008, the entire disclosure of
which is
hereby incorporated by reference, Compound B is the compound of example 345.
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Compound B can be prepared as described in International Application No.
PCT/US2008/063819.
Suitably, Compound B is in the form of free base.
The compounds of the invention may form a solvate which is understood to be a
complex of variable stoichiometry formed by a solute (in this invention,
Compound A or a
salt thereof and/or Compound B or a salt thereof) and a solvent. Such solvents
for the
purpose of the invention may not interfere with the biological activity of the
solute.
Examples of suitable solvents include, but are not limited to, water,
methanol, dimethyl
sulfoxide, ethanol and acetic acid. Suitably the solvent used is a
pharmaceutically
acceptable solvent. Examples of suitable pharmaceutically acceptable solvents
include,
without limitation, water, dimethyl sulfoxide, ethanol and acetic acid.
Suitably the solvent
used is water.
The pharmaceutically acceptable salts of the compounds of the invention are
readily prepared by those of skill in the art.
By the term "treating" and derivatives thereof as used herein, is meant
therapeutic
therapy. In reference to a particular condition, treating means: (1) to
ameliorate or
prevent the condition of one or more of the biological manifestations of the
condition, (2)
to interfere with (a) one or more points in the biological cascade that leads
to or is
responsible for the condition or (b) one or more of the biological
manifestations of the
condition, (3) to alleviate one or more of the symptoms, effects or side
effects associated
with the condition or treatment thereof, or (4) to slow the progression of the
condition or
one or more of the biological manifestations of the condition. Prophylactic
therapy is also
contemplated thereby. The skilled artisan will appreciate that "prevention" is
not an
absolute term. In medicine, "prevention" is understood to refer to the
prophylactic
administration of a drug to substantially diminish the likelihood or severity
of a condition or
biological manifestation thereof, or to delay the onset of such condition or
biological
manifestation thereof. Prophylactic therapy is appropriate, for example, when
a subject is
considered at high risk for developing cancer, such as when a subject has a
strong family
history of cancer or when a subject has been exposed to a carcinogen.
By the term "periodically administration" or variations thereof, is meant that
the
drug is not administered to the human with drug holidays. A drug holiday
(sometimes
also called a drug vacation, medication vacation, structured treatment
interruption or
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strategic treatment interruption) is when a patient stops taking a
medication(s) for a
period of time; anywhere from a few days to several months
As used herein, the term "effective amount" means that amount of a drug or
pharmaceutical agent that will elicit the biological or medical response of a
tissue, system,
animal or human that is being sought, for instance, by a researcher or
clinician.
Furthermore, the term "therapeutically effective amount" means any amount
which, as
compared to a corresponding subject who has not received such amount, results
in
improved treatment, healing, prevention, or amelioration of a disease,
disorder, or side
effect, or a decrease in the rate of advancement of a disease or disorder. The
term also
includes within its scope amounts effective to enhance normal physiological
function.
By the term "combination" and derivatives thereof, as used herein is meant
either
simultaneous administration or any manner of separate sequential
administration of a
therapeutically effective amount of Compound A, or a pharmaceutically
acceptable salt or
solvate thereof, and Compound B or a pharmaceutically acceptable salt thereof.
Preferably, if the administration is not simultaneous, the compounds are
administered in a
close time proximity to each other. Furthermore, it does not matter if the
compounds are
administered in the same dosage form, e.g. one compound may be administered
topically
and the other compound may be administered orally. Suitably, both compounds
are
administered orally.
By the term "combination kit" as used herein is meant the pharmaceutical
composition or compositions that are used to administer Compound A, or a
pharmaceutically acceptable salt or solvate thereof, and Compound B, or a
pharmaceutically acceptable salt thereof, according to the invention. When
both
compounds are administered simultaneously, the combination kit can contain
Compound
A, or a pharmaceutically acceptable salt or solvate thereof, and Compound B,
or a
pharmaceutically acceptable salt thereof, in a single pharmaceutical
composition, such as
a tablet, or in separate pharmaceutical compositions. When the compounds are
not
administered simultaneously, the combination kit will contain Compound A, or a
pharmaceutically acceptable salt or solvate thereof, and Compound B, or a
pharmaceutically acceptable salt thereof, in separate pharmaceutical
compositions. The
combination kit can comprise Compound A, or a pharmaceutically acceptable salt
or
solvate thereof, and Compound B, or a pharmaceutically acceptable salt
thereof, in
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separate pharmaceutical compositions in a single package or in separate
pharmaceutical
compositions in separate packages.
In one aspect there is provided a combination kit comprising the components:
Compound A, or a pharmaceutically acceptable salt or solvate thereof, in
association with
a pharmaceutically acceptable carrier; and
Compound B, or a pharmaceutically acceptable salt thereof, in association with
a
pharmaceutically acceptable carrier.
In one embodiment of the invention the combination kit comprises the following
components:
Compound A, or a pharmaceutically acceptable salt or solvate thereof, in
association with
a pharmaceutically acceptable carrier; and
Compound B, or a pharmaceutically acceptable salt thereof, in association with
a
pharmaceutically acceptable carrier,
wherein the components are provided in a form which is suitable for
sequential, separate
and/or simultaneous administration.
In one embodiment the combination kit comprises:
a first container comprising Compound A, or a pharmaceutically acceptable salt
or solvate
thereof, in association with a pharmaceutically acceptable carrier; and
a second container comprising Compound B, or a pharmaceutically acceptable
salt
thereof, in association with a pharmaceutically acceptable carrier, and a
container means
for containing said first and second containers.
The "combination kit" can also be provided by instruction, such as dosage and
administration instructions. Such dosage and administration instructions can
be of the
kind that is provided to a doctor, for example by a drug product label, or
they can be of
the kind that is provided by a doctor, such as instructions to a patient.
By the term "triple negative" breast cancer, as used herein is meant any
breast
cancer that does not express the genes for estrogen receptor (ER),
progesterone
receptor (PR) or Her2/neu. This subtype of breast cancer is clinically
characterised as
more aggressive and less responsive to standard treatment and associated
poorer overall
patient prognosis. It is diagnosed more frequently in younger women, women
with
BRCA1 mutations, and those belonging to African-American and Hispanic ethnic
groups,
and those having a recent birth.
A basal-like breast tumor is a subtype of aggressive breast cancer that has a
short
relapse time. African-American women that are premenopausal are at higher than
average risk to develop basal-like breast tumors, which are usually triple-
negative for
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estrogen, progesterone, and HER2 receptors. Basal-like breast tumors may be
high
grade and diagnosed at a late stage, requiring powerful chemotherapy regimens.
As used herein the term "Compound A2" means ---Compound A, or a
pharmaceutically acceptable salt or solvate thereof---.
As used herein the term "Compound B2" means ---Compound B, or a
pharmaceutically acceptable salt thereof---.
Suitably the combinations of this invention are administered within a
"specified
period".
By the term "specified period" and derivatives thereof, as used herein is
meant the
interval of time between the administration of one of Compound A2 and Compound
B2
and the other of Compound A2 and Compound B2. Unless otherwise defined, the
specified period can include simultaneous administration. Unless otherwise
defined the
specified period refers to administration of Compound A2 and Compound B2
during a
single day.
Suitably, if the compounds are administered within a "specified period" and
not
administered simultaneously, they are both administered within about 24 hours
of each
other - in this case, the specified period will be about 24 hours; suitably
they will both be
administered within about 12 hours of each other - in this case, the specified
period will
be about 12 hours; suitably they will both be administered within about 11
hours of each
other - in this case, the specified period will be about 11 hours; suitably
they will both be
administered within about 10 hours of each other - in this case, the specified
period will
be about 10 hours; suitably they will both be administered within about 9
hours of each
other - in this case, the specified period will be about 9 hours; suitably
they will both be
administered within about 8 hours of each other - in this case, the specified
period will be
about 8 hours; suitably they will both be administered within about 7 hours of
each other -
in this case, the specified period will be about 7 hours; suitably they will
both be
administered within about 6 hours of each other - in this case, the specified
period will be
about 6 hours; suitably they will both be administered within about 5 hours of
each other -
in this case, the specified period will be about 5 hours; suitably they will
both be
administered within about 4 hours of each other - in this case, the specified
period will be
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about 4 hours; suitably they will both be administered within about 3 hours of
each other -
in this case, the specified period will be about 3 hours; suitably they will
be administered
within about 2 hours of each other - in this case, the specified period will
be about 2
hours; suitably they will both be administered within about 1 hour of each
other - in this
case, the specified period will be about 1 hour. As used herein, the
administration of
Compound A2 and Compound B2 in less than about 45 minutes apart is considered
simultaneous administration.
Suitably, when the combination of the invention is administered for a
"specified
period", the compounds will be co-administered for a "duration of time".
By the term "duration of time" and derivatives thereof, as used herein is
meant
that both compounds of the invention are administered for an indicated number
of
consecutive days. Unless otherwise defined, the number of consecutive days
does not
have to commence with the start of treatment or terminate with the end of
treatment, it is
only required that the number of consecutive days occur at some point during
the course
of treatment.
Regarding "specified period" administration:
Suitably, both compounds will be administered within a specified period for at
least one day - in this case, the duration of time will be at least one day;
suitably, during
the course to treatment, both compounds will be administered within a
specified period for
at least 3 consecutive days - in this case, the duration of time will be at
least 3 days;
suitably, during the course to treatment, both compounds will be administered
within a
specified period for at least 5 consecutive days - in this case, the duration
of time will be
at least 5 days; suitably, during the course to treatment, both compounds will
be
administered within a specified period for at least 7 consecutive days - in
this case, the
duration of time will be at least 7 days; suitably, during the course to
treatment, both
compounds will be administered within a specified period for at least 14
consecutive days
- in this case, the duration of time will be at least 14 days; suitably,
during the course to
treatment, both compounds will be administered within a specified period for
at least 30
consecutive days - in this case, the duration of time will be at least 30
days.
Suitably, if the compounds are not administered during a "specified period",
they
are administered sequentially. By the term "sequential administration", and
derivates
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thereof, as used herein is meant that one of Compound A2 and Compound B2 is
administered once a day for two or more consecutive days and the other of
Compound A2
and Compound B2 is subsequently administered once a day for two or more
consecutive
days. Also, contemplated herein is a drug holiday utilized between the
sequential
administration of one of Compound A2 and Compound B2 and the other of Compound
A2
and Compound B2. As used herein, a drug holiday is a period of days after the
sequential administration of one of Compound A2 and Compound B2 and before the
administration of the other of Compound A2 and Compound B2 where neither
Compound
A2 nor Compound B2 is administered. Suitably the drug holiday will be a period
of days
selected from: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days,
9 days, 10
days, 11 days, 12 days, 13 days and 14 days.
Regarding sequential administration:
Suitably, one of Compound A2 and Compound B2 is administered for from 2 to 30
consecutive days, followed by an optional drug holiday, followed by
administration of the
other of Compound A2 and Compound B2 for from 2 to 30 consecutive days.
Suitably,
one of Compound A2 and Compound B2 is administered for from 2 to 21
consecutive
days, followed by an optional drug holiday, followed by administration of the
other of
Compound A2 and Compound B2 for from 2 to 21 consecutive days. Suitably, one
of
Compound A2 and Compound B2 is administered for from 2 to 14 consecutive days,
followed by a drug holiday of from 1 to 14 days, followed by administration of
the other of
Compound A2 and Compound B2 for from 2 to 14 consecutive days. Suitably, one
of
Compound A2 and Compound B2 is administered for from 3 to 7 consecutive days,
followed by a drug holiday of from 3 to 10 days, followed by administration of
the other of
Compound A2 and Compound B2 for from 3 to 7 consecutive days.
Suitably, Compound B2 will be administered first in the sequence, followed by
an
optional drug holiday, followed by administration of Compound A2. Suitably,
Compound
B2 is administered for from 3 to 21 consecutive days, followed by an optional
drug
holiday, followed by administration of Compound A2 for from 3 to 21
consecutive days.
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Suitably, Compound B2 is administered for from 3 to 21 consecutive days,
followed by a
drug holiday of from 1 to 14 days, followed by administration of Compound A2
for from 3
to 21 consecutive days. Suitably, Compound B2 is administered for from 3 to 21
consecutive days, followed by a drug holiday of from 3 to 14 days, followed by
administration of Compound A2 for from 3 to 21 consecutive days. Suitably,
Compound
B2 is administered for 21 consecutive days, followed by an optional drug
holiday, followed
by administration of Compound A2 for 14 consecutive days. Suitably, Compound
B2 is
administered for 14 consecutive days, followed by a drug holiday of from 1 to
14 days,
followed by administration of Compound A2 for 14 consecutive days. Suitably,
Compound B2 is administered for 7 consecutive days, followed by a drug holiday
of from
3 to 10 days, followed by administration of Compound A2 for 7 consecutive
days.
Suitably, Compound B2 is administered for 3 consecutive days, followed by a
drug
holiday of from 3 to 14 days, followed by administration of Compound A2 for 7
consecutive days. Suitably, Compound B2 is administered for 3 consecutive
days,
followed by a drug holiday of from 3 to 10 days, followed by administration of
Compound
A2 for 3 consecutive days.
It is understood that a "specified period" administration and a "sequential"
administration can be followed by repeat dosing or can be followed by an
alternate dosing
protocol, and a drug holiday may precede the repeat dosing or alternate dosing
protocol.
Suitably, the amount of Compound A2 administered as part of the combination
according to the present invention will be an amount selected from about
0.125mg to
about 10mg; suitably, the amount will be selected from about 0.25mg to about
9mg;
suitably, the amount will be selected from about 0.25mg to about 8mg;
suitably, the
amount will be selected from about 0.5mg to about 8mg; suitably, the amount
will be
selected from about 0.5mg to about 7mg; suitably, the amount will be selected
from about
1 mg to about 7mg; suitably, the amount will be about 5mg. Accordingly, the
amount of
Compound A administered as part of the combination according to the present
invention
will be an amount selected from about 0.125mg to about 10 mg. For example, the
amount of Compound A2 administered as part of the combination according to the
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present invention can be 0.125mg, 0.25mg, 0.5mg, 0.75mg, 1 mg, 1.5mg, 2mg,
2.5mg,
3mg, 3.5mg, 4mg, 4.5mg, 5mg, 5.5mg, 6mg, 6.5mg, 7mg, 7.5mg, 8mg, 8.5mg, 9mg,
9.5mg, 10mg.
Suitably, the amount of Compound B2 administered as part of the combination
according to the present invention will be an amount selected from about
0.25mg to about
75mg; suitably, the amount will be selected from about 0.5mg to about 50mg;
suitably,
the amount will be selected from about 1 mg to about 25mg; suitably, the
amount will be
selected from about 2mg to about 20mg; suitably, the amount will be selected
from about
4mg to about 16mg; suitably, the amount will be selected from about 6mg to
about 12mg;
suitably, the amount will be about 10mg. Accordingly, the amount of Compound
B2
administered as part of the combination according to the present invention
will be an
amount selected from about 0.5mg to about 50mg. For example, the amount of
Compound B2 administered as part of the combination according to the present
invention
can be 0.5mg, 1 mg, 2mg, 3mg, 4mg, 5mg, 6mg, 7mg, 8mg, 9mg, 10mg, 11 mg, 12mg,
13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 20mg, 21 mg, 22mg, 23mg, 25mg, 26mg, 27mg,
28mg, 29mg, 30mg, 35mg, 40mg, 45mg, or 50mg.
As used herein, all amounts specified for Compound A2 and Compound B2 are
indicated as the administered amount of free or unsalted and unsolvated
compound per
dose.
The method of the present invention may also be employed with other
therapeutic
methods of cancer treatment.
While it is possible that, for use in therapy, therapeutically effective
amounts of the
combinations of the present invention may be administered as the raw chemical,
it is
preferable to present the combinations as a pharmaceutical composition or
compositions.
Accordingly, the invention further provides pharmaceutical compositions, which
include
Compound A2 and/or Compound B2, and one or more pharmaceutically acceptable
carriers. The combinations of the present invention are as described above.
The
carrier(s) must be acceptable in the sense of being compatible with the other
ingredients
of the formulation, capable of pharmaceutical formulation, and not deleterious
to the
recipient thereof. In accordance with another aspect of the invention there is
also
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provided a process for the preparation of a pharmaceutical formulation
including admixing
Compound A2 and/or Compound B2 with one or more pharmaceutically acceptable
carriers. As indicated above, such elements of the pharmaceutical combination
utilized
may be presented in separate pharmaceutical compositions or formulated
together in one
pharmaceutical formulation.
Pharmaceutical formulations may be presented in unit dose forms containing a
predetermined amount of active ingredient per unit dose. As is known to those
skilled in
the art, the amount of active ingredient per dose will depend on the condition
being
treated, the route of administration and the age, weight and condition of the
patient.
Preferred unit dosage formulations are those containing a daily dose or sub-
dose, or an
appropriate fraction thereof, of an active ingredient. Furthermore, such
pharmaceutical
formulations may be prepared by any of the methods well known in the pharmacy
art.
Compound A2 and Compound B2 may be administered by any appropriate
route. Suitable routes include oral, rectal, nasal, topical (including buccal
and sublingual),
vaginal, and parenteral (including subcutaneous, intramuscular, intravenous,
intradermal,
intrathecal, and epidural). It will be appreciated that the preferred route
may vary with, for
example, the condition of the recipient of the combination and the cancer to
be treated. It
will also be appreciated that each of the agents administered may be
administered by the
same or different routes and that Compound A2 and Compound B2 may be
compounded
together in a pharmaceutical composition/formulation.
The compounds or combinations of the current invention are incorporated
into convenient dosage forms such as capsules, tablets, or injectable
preparations. Solid
or liquid pharmaceutical carriers are employed. Solid carriers include,
starch, lactose,
calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin,
acacia,
magnesium stearate, and stearic acid. Liquid carriers include syrup, peanut
oil, olive oil,
saline, and water. Similarly, the carrier may include a prolonged release
material, such
as glyceryl monostearate or glyceryl distearate, alone or with a wax. The
amount of solid
carrier varies widely but, preferably, will be from about 25 mg to about 1 g
per dosage
unit. When a liquid carrier is used, the preparation will suitably be in the
form of a syrup,
elixir, emulsion, soft gelatin capsule, sterile injectable liquid such as an
ampoule, or an
aqueous or nonaqueous liquid suspension.
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For instance, for oral administration in the form of a tablet or capsule, the
active
drug component can be combined with an oral, non-toxic pharmaceutically
acceptable
inert carrier such as ethanol, glycerol, water and the like. Powders are
prepared by
comminuting the compound to a suitable fine size and mixing with a similarly
comminuted
pharmaceutical carrier such as an edible carbohydrate, as, for example, starch
or
mannitol. Flavoring, preservative, dispersing and coloring agent can also be
present.
It should be understood that in addition to the ingredients mentioned above,
the
formulations may include other agents conventional in the art having regard to
the type of
formulation in question, for example those suitable for oral administration
may include
flavoring agents.
As indicated, therapeutically effective amounts of the combinations of the
invention (Compound A2 in combination with Compound B2) are administered to a
human. Typically, the therapeutically effective amount of the administered
agents of the
present invention will depend upon a number of factors including, for example,
the age
and weight of the subject, the precise condition requiring treatment, the
severity of the
condition, the nature of the formulation, and the route of administration.
Ultimately, the
therapeutically effective amount will be at the discretion of the attendant
physician.
The combinations of the present invention are tested for efficacy,
advantageous
and synergistic properties according to known procedures. Suitably, the
combinations of
the invention are tested for efficacy, advantageous and synergistic properties
generally
according to the following combination cell proliferation assays. Cells are
plated in 96 or
384-well plates in culture media appropriate for each cell type, supplemented
with 10%
FBS and 1 % penicillin/streptomycin, and incubated overnight at 37 C, 5% CO2.
Cells are
treated in a grid manner with dilution of Compound A2 (10 dilutions, including
no
compound, of 3-fold dilutions starting from 0.250-20 .tM depending of
compound) and
also treated with Compound B2 (10 dilutions, including no compound, of 3-fold
dilutions
starting from 0.150-20 .tM depending of compound) and incubated as above for a
further
72 hours. In some instances compounds are added in a staggered manner and
incubation time can be extended up to 7days. Cell growth is measured using
CellTiter-
Glo reagent according to the manufacturer's protocol and signals are read on
a
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PerkinElmer EnVisionTM reader set for luminescence mode with a 0.5-second
read. Data
are analyzed as described below.
Results are expressed as a percentage of the t=0 value and plotted against
compound(s) concentration. The t=0 value is normalized to 100% and represents
the
number of cells present at the time of compound addition. The cellular
response is
determined for each compound and/or compound combination using a 4- or 6-
parameter
curve fit of cell viability against concentration using the IDBS XLfit plug-in
for Microsoft
Excel software and determining the concentration required for 50% inhibition
of cell
growth (glC5o). Background correction is made by subtraction of values from
wells
containing no cells. For each drug combination a Combination Index (CI),
Excess Over
Highest Single Agent (EOHSA) and Excess Over Bliss (EOBliss) are calculated
according
to known methods such as described in Chou and Talalay (1984) Advances in
Enzyme
Regulation, 22, 37 to 55; and Berenbaum, MC (1981) Adv. Cancer Research, 35,
269-
335.
Because the combinations of the present invention are active in the above
assays
they exhibit advantageous therapeutic utility in treating cancer.
Suitably, the present invention relates to a method for treating or lessening
the
severity of a cancer selected from: brain (gliomas), glioblastomas, Bannayan-
Zonana
syndrome, Cowden disease, Lhermitte-Duclos disease, breast, inflammatory
breast
cancer, Wilm's tumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma,
medulloblastoma, colon, head and neck, kidney, lung, liver, melanoma, ovarian,
pancreatic, prostate, sarcoma, osteosarcoma, giant cell tumor of bone,
thyroid,
Lymphoblastic T cell leukemia, Chronic myelogenous leukemia, Chronic
lymphocytic leukemia, Hairy-cell leukemia, acute lymphoblastic leukemia, acute
myelogenous leukemia, Chronic neutrophilic leukemia, Acute lymphoblastic T
cell
leukemia, Plasmacytoma, Immunoblastic large cell leukemia, Mantle cell
leukemia,
Multiple myeloma Megakaryoblastic leukemia, multiple myeloma, acute
megakaryocytic
leukemia, promyelocytic leukemia, Erythroleukemia,
malignant lymphoma, hodgkins lymphoma, non-hodgkins lymphoma,
lymphoblastic T cell lymphoma, Burkitt's lymphoma, follicular lymphoma,
neuroblastoma, bladder cancer, urothelial cancer, lung cancer, vulval cancer,
cervical cancer, endometrial cancer, renal cancer, mesothelioma, esophageal
cancer,
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salivary gland cancer, hepatocellular cancer, gastric cancer, nasopharangeal
cancer,
buccal cancer, cancer of the mouth, GIST (gastrointestinal stromal tumor) and
testicular
cancer.
Suitably, the present invention relates to a method for treating or lessening
the
severity of a cancer selected from: brain (gliomas), glioblastomas, Bannayan-
Zonana
syndrome, Cowden disease, Lhermitte-Duclos disease, breast, colon, head and
neck,
kidney, lung, liver, melanoma, ovarian, pancreatic, prostate, sarcoma and
thyroid.
Suitably, the present invention relates to a method for treating or lessening
the
severity of a cancer selected from ovarian, liver, colon, breast, pancreatic
and prostate.
Suitably, the present invention relates to a method for treating or lessening
the
severity of a cancer selected from breast, liver, lung, pancreatic, and colon.
Suitably, the present invention relates to a method of treating or lessening
the
severity of a cancer that is either wild type or mutant for certain
biomarker(s).
The term "wild type" as is understood in the art refers to a polypeptide or
polynucleotide sequence that occurs in a native population without genetic
modification.
As is also understood in the art, a "mutant" includes a polypeptide or
polynucleotide
sequence having at least one modification to an amino acid or nucleic acid
compared to
the corresponding amino acid or nucleic acid found in a wild type polypeptide
or
polynucleotide, respectively. Included in the term mutant is Single Nucleotide
Polymorphism (SNP) where a single base pair distinction exists in the sequence
of a
nucleic acid strand compared to the most prevalently found (wild type) nucleic
acid
strand.
Cancers that are either wild type or mutant for biomarker(s) and either wild
type or
mutant for P13K/Pten are identified by known methods.
V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog, also known as KRAS, is a
protein which in humans is encoded by the KRAS gene. Like other members of the
Ras
family, the KRAS protein is a GTPase and is an early player in many signal
transduction
pathways. KRAS is usually tethered to cell membranes because of the presence
of an
isoprenyl group on its C-terminus. When mutated, KRAS is an oncogene. The
protein
product of the normal KRAS gene performs an essential function in normal
tissue
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signaling, and the mutation of a KRAS gene is an essential step in the
development of
many cancers.
The N-ras oncogene is a member of the RAS gene family. It is mapped on
chromosome 1, and it is activated in HL60, a promyelocytic leukemia line. The
order of
nearby genes is as follows: cen--CD2--NGFB--NRAS--tel. The mammalian ras gene
family consists of the harvey and kirsten ras genes (c-Hrasl and c-Kras2), an
inactive
pseudogene of each (c-Hras2 and c-Krasl) and the N-ras gene. They differ
significantly
only in the C-terminal 40 amino acids. These ras genes have GTP/GDP binding
and
GTPase activity, and their normal function may be as G-like regulatory
proteins involved
in the normal control of cell growth. Mutations which change amino acid
residues 12, 13
or 61 activate the potential of N-ras to transform cultured cells and are
implicated in a
variety of human tumors. The N-ras gene specifies two main transcripts of 2Kb
and
4.3Kb. The difference between the two transcripts is a simple extension
through the
termination site of the 2Kb transcript. The N-ras gene consists of seven exons
(-I, I, II, III,
IV, V, VI). The smaller 2Kb transcript contains the Vla exon, and the larger
4.3Kb
transcript contains the VIb exon which is just a longer form of the Vla exon.
Both
transcripts encode identical proteins as they differ only the 3' untranslated
region. The
sequence of the shorter 2Kb transcript is presented here. The 4.3 Kb
transcript sequence
is not available.
Wild type or mutant Ras/Raf or P13K/PTEN tumor cells can be identified by DNA
amplification and sequencing techniques, DNA and RNA detection techniques,
including,
but not limited to Northern and Southern blot, respectively, and/or various
biochip and
array technologies. This can include cytogenetic aberrations and transcript
abundance.
Wild type and mutant polypeptides can be detected by a variety of techniques
including,
but not limited to immunodiagnostic techniques such as ELISA, Western blot or
imunocyto chemistry.
This invention provides a combination comprising N-{3-[3-cyclopropyl-5-[(2-
fluoro-
4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-
d]pyrimidin-
1(2H)-yl]phenyl}acetamide, or a pharmaceutically acceptable salt or solvate
thereof, and
2,4-difl uoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-q uin ol inyl]-3-
pyridinyl}benzenesulfonamide, or a pharmaceutically acceptable salt thereof.
This invention also provides for a combination comprising N-{3-[3-cyclopropyl-
5-
[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl -2,4,7-trioxo-3,4,6,7-
tetrahydropyrido[4,3-
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d]pyrimidin-1(2H)-yl]phenyl}acetamide, or a pharmaceutically acceptable salt
or solvate
thereof, and 2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-
3-
pyridinyl}benzenesulfonamide, or a pharmaceutically acceptable salt thereof,
for use in
therapy.
This invention also provides for a combination comprising N-{3-[3-cyclopropyl-
5-
[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl -2,4,7-trioxo-3,4,6,7-
tetrahydropyrido[4,3-
d]pyrimidin-1(2H)-yl]phenyl}acetamide, or a pharmaceutically acceptable salt
or solvate
thereof, and 2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-
3-
pyridinyl}benzenesulfonamide, or a pharmaceutically acceptable salt thereof,
for use in
treating cancer.
This invention also provides a pharmaceutical composition comprising a
combination of N-{3-[3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-di
methyl-2,4,7-
trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl]phenyl}acetamide, or
a
pharmaceutically acceptable salt or solvate thereof, and 2,4-difluoro-N-{2-
(methyloxy)-5-
[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide, or a
pharmaceutically
acceptable salt thereof.
This invention also provides a combination kit comprising N-{3-[3-cyclopropyl-
5-
[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl -2,4,7-trioxo-3,4,6,7-
tetrahydropyrido[4,3-
d]pyrimidin-1(2H)-yl]phenyl}acetamide, or a pharmaceutically acceptable salt
or solvate
thereof, and 2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-
3-
pyridinyl}benzenesulfonamide, or a pharmaceutically acceptable salt thereof.
This invention also provides for the use of a combination comprising N-{3-[3-
cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6, 8-d imethyl-2,4, 7-trioxo-3,4,
6,7-
tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl]phenyl}acetamide, or a
pharmaceutically
acceptable salt or solvate thereof, and 2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-
pyridazinyl)-
6-quinolinyl]-3-pyridinyl}benzenesulfonamide, or a pharmaceutically acceptable
salt
thereof, in the manufacture of a medicament.
This invention also provides for the use of a combination comprising N-{3-[3-
cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6, 8-d imethyl-2,4, 7-trioxo-3,4,
6,7-
tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl]phenyl}acetamide, or a
pharmaceutically
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acceptable salt or solvate thereof, and 2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-
pyridazinyl)-
6-quinolinyl]-3-pyridinyl}benzenesulfonamide, or a pharmaceutically acceptable
salt
thereof, in the manufacture of a medicament to treat cancer.
This invention also provides a method of treating cancer which comprises
administering a combination of N-{3-[3-cyclopropyl-5-[(2-fluoro-4-
iodophenyl)amino]-6,8-
dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-
yl]phenyl}acetamide,
or a pharmaceutically acceptable salt or solvate thereof, and 2,4-difluoro-N-
{2-
(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide,
or a
pharmaceutically acceptable salt thereof, to a subject in need thereof.
This invention also relates to a method of treating cancer, which comprises
administsering a combination of N-{3-[3-cyclopropyl-5-[(2-fluoro-4-
iodophenyl)amino]-6,8-
dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-
yl]phenyl}acetamide,
or a pharmaceutically acceptable salt or solvate thereof, and 2,4-difluoro-N-
{2-
(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide,
or a
pharmaceutically acceptable salt thereof, to a subject in need thereof,
wherein the
amount of N-{3-[3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-
2,4,7-trioxo-
3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl]phenyl}acetamide, or a
pharmaceutically
acceptable salt or solvate thereof is selected from about 0.5 mg to about 3 mg
and the
amount of 2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-
pyridinyl}benzenesulfonamide, or a pharmaceutically acceptable salt or solvate
thereof is
selected from about 0.5 mg to about 3 mg.
The following examples are intended for illustration only and are not intended
to
limit the scope of the invention in any way.
Experimental Details
Preparation of MEK inhibitors
MEK inhibitors which are suitable for use in the present combinations,
particularly
N-{3-[3-cyclopropyl-5-(2-fluoro-4-iodo-phenylamino)6,8-dimethy;-2,4,7-trioxo-
3,4,6,7-
tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamide dimethyl sulfoxide,
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F
~ I
0 HN
CH
N N' s
O N O
CHs
H3C N
can be prepared according to International Patent Publication No.
W02005/121142.
P13K inhibitors which are suitable for use in the present combinations,
particularly
2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-
pyridinyl}benzenesulfonamide
N,- N
N
F 0 0 /
SAN \ I \ \
H
F N
can be prepared according to International Patent Publication No. W008/144463
(Example 345)
Compound A as described in the Experimental section refers to the dimethyl
sulfoxide
solvate of N-{3-[3-cyclopropyl-5-(2-fluoro-4-iodo-phenylamino)6,8-dimethy;-
2,4,7-trioxo-
3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamide.
In vitro cell growth inhibition and apoptosis induction by Compound A,
Compound
B and their combination in tumor cell lines
Study #1. Colon, Lung and Pancreatic Cancer Cell Lines
Experimental Preparation(s)
Combination drug tests with Compounds A and B were conducted using a panel
of cell lines from human colon cancers (n = 26), lung cancers (n = 14) and
pancreatic
cancers (n = 6) (Table 1). Cell lines were purchased commercially [from ATCC
(Manassas, VA, USA) or DSMZ (Braunschweig, Germany)] and grown in RPMI-1640
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supplemented with 2 mM glutamine, 1 mM sodium pyruvate and 10% fetal bovine
serum
(except for Capan-1 and HuP-T4 which were grown with 20% fetal bovine serum)
and
maintained at 37 C and 5% CO2 in a humid incubator.
Experimental Protocol(s)
Fixed Ratio Drug Combination Assay
The dilution design of the Fixed Ratio Drug Combination Assay can be seen in
Figure 1. First, the test compounds were prepared as 10 mM stocks in 100%
dimethyl
sulfoxide (DMSO). Further dilutions of the compounds were made with DMSO. The
first
test compound (designated as Compound A) is diluted horizontally in a 96 well
microtiter
plate in rows B-E using a 3-fold dilution series for 10 dilution points. A
second test
compound (designated as Compound B) is diluted horizontally in a separate 96
well
microtiter plate in rows D-G using a 3-fold dilution series for 10 dilution
points. The two
compounds are combined using equal volumes from each drug plate into cell
culture
media. This results in a 1:50 dilution of the drugs in the cell culture media.
Compound A
is individually titrated in rows B and C, while only Compound B is dosed in
rows F and G
of the plate. An additional 1:10 dilution of the drugs is performed in cell
culture media
prior to addition to the cells. Drug addition to the cells results in a
further 1:2 dilution of
drugs. The total dilution of the drug plate to the cells is 1:1000. The final
dosing
concentration range for Compound B was 0.008 - 150.0 nM and was 0.013 - 250.0
nM
for Compound A. The positive control consists of culture media with DMSO at
0.1% and
cells and no drug. The negative control consists of culture media with DMSO at
0.1 %.
solution.
Assays were performed in 96 well microtiter plates with appropriate seeding
densities estimated from previous studies of each cell line. Following dosing,
the cell
lines are incubated at 37 C, 5% CO2 in humid air for 72 hours. Cell
proliferation was
measured using the CeIlTiter Glo (Promega Corporation, Madison, WI, USA)
reagent
according to the manufacturer's protocol. The plates are treated with
CeIlTiter Glo
solution and are analyzed for RLU (relative light units) using a Molecular
Devices
SpectraMax M5 (Sunnyvale, CA, USA) plate reader.
Data Analysis
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Three independent metrics were used to analyze the combinatorial effects on
growth inhibition of Compound B and Compound A.
1. Excess over Highest Single Agent (EOHSA)- One standard criterion for
measuring drug combinatorial effects is analyzing the effects on cell growth
inhibition in absolute terms. In this case, the combination of drugs is
compared to
the more responsive of the two individual treatments (single agent). For each
combination experiment, the percent effect relative to the highest single
agent for
each dose along the curve is generated. This measure of "Excess of Highest
Single Agent (EOHSA)" is one of the criteria used for evaluating synergy of
drug
combinations. (Borisy AA Elliott PJ, Hurst NW, Lee MS, Lehar J, Price ER,
Serbedzija G,Zimmermann GR, Foley MA, Stockwell BR, Keith CT. Systematic
discovery of multicomponent therapeutics. Proc Natl Acad Sci U S A. 2003 Jun
24;100(13):7977-82)
2. Bliss synergy- A second criterion often used to determine combination
synergy is
evaluating the excess inhibition over Bliss independence or "additivity"
(Bliss, C.I,
Mexico, DF, The Toxicity of Poisons Applied Jointly. Annals of Applied Biology
1939, Vol 26, Issue 3, August 1939). The model assumes a combined response of
the two compounds independently using the following:
Score = Ea + Eb - (Ea * Eb
Where Ea is the effect (or percent inhibition) of compound A and Eb is the
effect of
compound B. The resulting effect of the combination of the two compounds is
compared to their predicted additivity by Bliss and a synergy score is
generated
for each dose along the response curve.
3. Combination Index (Cl)- A third criterion for evaluation of synergy is
Combination
Index (CI) derived from the Chou and Talalay (Chou TC, Talalay P. Quantitative
analysis of dose-effect relationships: the combined effects of multiple drugs
or
enzyme inhibitors. Adv Enzyme Regul.1984;22:27-55). The following equation is
a
model used for compounds that behave with different mechanisms of action
(mutually non-exclusive formula).
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Combination Index = D ina:b + Dbina:b + (D ina:bXDbina:b)
IC50(Q) IC50(b) IC50(a) ICso(b)
The lower the Cl the more synergy the combination potentially has. A Cl
greater
than 1 suggests that the combination being studied may be antagonistic. Cl
scores are also generated for inhibitory concentrations of 25%(l C25) and 75%
(IC75) by replacing the IC50 in the formula above for each compound with the
respective inhibitory concentration.
The percent intensity values were used in model 205 of XLfit in Microsoft
Excel to
calculate gIC5o values using a 4 parameter logistical fit. The midpoint of the
growth
window (the gIC5o) falls half way between the number of cells at the time of
compound
addition (T=0) and the growth of control cells treated with DMSO at 72 hrs.
The number
of cells at time zero (To) is divided from the intensity value at the bottom
of the response
curve (Ymin) to generate a measure for cell death (Ymin/To). A value below 1
for Ymin/To
indicates stronger potency with the treatment when compared to higher values.
For EOHSA and Bliss, a synergy score must be seen in both technical
replications
within an experiment to make an appropriate designation (synergy, modest
synergy, etc).
Each combination experiment contains a replicate for the two compounds as
single
agents as well as a technical replicate for the combination.
Synergy scores for EOHSA and Bliss, at extremely low concentrations, (e.g.
Dose
1, dose 2) are subject to higher variation and generally excluded from the
analysis.
Conversely, synergy scores at the highest concentrations (Dose 10), far
outside of the
therapeutic dosing range, are generally excluded from analysis since the
effects observed
are more susceptible to off-target events.
For EOHSA and Bliss Synergy measures, a score is generated for each dose
along the response curve. Scores were categorized as being `Antagonistic' (< -
10),
`Additive' (-10 - 10), `Modest Synergy' (10 - 20) or `Synergistic' (> 20).
These scores
reflect the percentage over the highest agent or percentage greater than Bliss
additivity,
depending on which model is being interpreted.
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For the Combination Index, the lower the Cl, the more synergy the combination
potentially has. Scores between 0 and 0.7 were considered to be synergistic,
while
scores between 0.7 and 0.9 were considered to be modest synergy. All other
scores did
not indicate synergy for the Combination index.
For those cell lines that never reached an inhibitory concentration of 25% for
1 of
the compounds in the combination, a Cl value cannot be calculated and `NA' was
listed
for the Cl.
Cell Line Mutation Data
Mutation data was collated for the status for the KRAS gene. The data source
is
the cancer cell line mutation screening data published as part of the Catolog
of Somatic
Mutations in Cancer database (COSMIC) (Bamford S. et al. Br. J. Cancer. 2004.
91:355-
58). In order to ensure that the identity of the cell lines used in the
proliferation assay
matched that in the COSMIC database, a genotype comparison was done between
those
cell lines in the sensitivity screen and those in COSMIC. Specifically, this
entailed:
1. Calculating the genotypes for each cell line using the Affymetrix 500K `SNP
Chip' (Affymetrix, Inc., Sunnyvale, CA) and the RLMM algorithm (Rabbee &
Speed, Bioinformatics, 2006. 22: 7-12).
2. Identifying the genotype matches of each cell line to those pre-calculated
for
each cell line having mutation profiles in COSMIC.
3. Assigning mutation status for each cell line in based upon the genotype
matches.
Results
A comprehensive categorization of the degree of synergy was done for each cell
line
treated with the combination of the P13K inhibitor Compound B and MEK
inhibitor
Compound A, Cell lines were considered to have synergy when at least one
metric was
scored as synergistic. Synergy data for Colon, Pancreatic, and Lung celllines
is
presented in Table 1-4. Data for pancreatic cell line calculations can be seen
in Appendix
A Tables 7-9.
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Table 1. Scores Panel of pancreatic, colon and lung cell lines used in
combination studies.
Cell Line Organ Site Diagnosis/Histology KRAS mutation status
NCI-H747 Colon Adenocarcinoma G13D
LS-1034 Colon Adenocarcinoma A146T
SW948 Colon Adenocarcinoma Q61 L
LS-174T Colon Adenocarcinoma G12D
SW116 Colon Adenocarcinoma G12A
T84 Colon Carcinoma G13D
Colo 201 Colon Adenocarcinoma WT
SW403 Colon Carcinoma G12V
DLD-1 Colon Carcinoma G13D
Colo 205 Colon Adenocarcinoma G12V
Colo320 HSR Colon Adenocarcinoma WT
SW620 Colon Adenocarcinoma G12V
NCI-H508 Colon Adenocarcinoma WT
Colo 320DM Colon Adenocarcinoma Unavail
SW837 Colon Adenocarcinoma G12C
KM-12 Colon Adenocarcinoma WT
WiDr Colon Adenocarcinoma WT
ileocecal colorectal G13D
HCT-8 Colon adenocarcinoma
RKO Colon Carcinoma WT
HT-29 Colon Carcinoma WT
SW480 Colon Adenocarcinoma G12V
HCT-15 Colon Adenocarcinoma G13D
HCT-116 Colon Carcinoma G13D
SW48 Colon Adenocarcinoma WT
SW1417 Colon Adenocarcinoma WT
HCC2998 Colon Carcinoma A146T
Calu 6 Lung Adenocarcinoma Q61 K
SK-MES-1 Lung Squamous cell carcinoma WT
Alveoloar basal epithelial- G12S
A549 Lung squamous
NCI-H2170 Lung Squamous cell carcinoma WT
NCI-H2228 Lung Adenocarcinoma WT
NCI-H23 Lung Adenocarcinoma WT
NCI-H1792 Lung Adenocarcinoma G12C
NCI-H358 Lung Branch io-alveolar G12C
NCI-H2122 Lung Adenocarcinoma G12C
NCI-H520 Lung Squamous cell carcinoma WT
NCI-H1299 Lung Non-small cell lung cancer WT
NCI-H1563 Lung Adenocarcinoma WT
NCI-H460 Lung Large cell carcinoma Q61 H
NCI-H2030 Lung Adenocarcinoma G12C
BxPC3 Pancreas Adenocarcinoma WT
SW1990 Pancreas Adenocarcinoma G12D
YAPC Pancreas Carcinoma G12V
MiaPaCa Pancreas Carcinoma G12C
Capan-1 Pancreas Adenocarcinoma G12V
HuP-T4 Pancreas Carcinoma G12V
Table 1 key
Cell Line = Cell line name
Organ Site = Organ from which cells were derived
Diagnosis/Histology = Pathological diagnosis of tissue
KRAS = Mutation status; WT = Wild Type
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Table 2. Basic measures and Synergy calls for each of the Colon cell lines.
Cell Line g1C50(nM) Ymin Ymin/To EOHSA BLISS Comb
Index
Colo201 0.56 4.04 0.22 Modest Additive Modest
... ................................................
................................................ ----____
............................................... ..Synergy......... Synergy
Colo205 0.67 -0.41 -0.06 Modest Additive Synergy
Synergy
Colo320DM 7.35 15.65 1.55 Modest Modest N/A
._SynergYSynergy
Colo320HSR 53.62 22.25 4.65 Antagonism Additive N/A
DLD1 7.77 12.21 1.71 Synergy Synergy Synergy
HCC2998 10.78 8.78 0.97 Synergy Synergy Synergy
HCT116 7.50 6.31 1.24 Synergy Synergy Modest
Synergy
HCT15 5.46 10.14 1.19 Synergy Modest N/A
Synergy----
HCT8 23.70 5.64 0.76 Synergy Modest Synergy
Synergy
HT29 0.59 1.70 0.19 Synergy Modest Synergy
Synergy----
KM12 21.95 8.21 0.61 Synergy Synergy Synergy
... .............................................
................................................ ......... _........ .........
......... ......... .............................................
LS1034 12.26 7.20 0.35 Modest Antagonism Modest
Synergy_-- Synergy
LS174T 46.60 4.02 0.19 Modest Antagonism N/A
_Synergy.....
NCIH508 0.08 1.90 0.07 Modest Additive Synergy
Synergy
NCIH747 2.59 4.65 0.14 Modest Additive Synergy
----.-Synergy
.........
RKO 25.52 1.42 0.15 Synergy Synergy Synergy
SW116 #N/A 11.25 0.18 Synergy Synergy Synergy
SW1417 1.09 21.32 1.21 Synergy Additive Synergy
SW403 1.04 1.51 0.05 Synergy Additive Synergy
... .................................................
............................................... ......... - .__.. .........
.................. ..................
.............................................
SW48 0.68 -0.30 -0.02 Synergy Modest Synergy
Synergy
SW480 1.35 17.03 0.67 Synergy Additive Synergy
SW620 6.95 14.69 2.12 Synergy Synergy N/A
... ..................................................
............................................... ......... __
............................................ ......... .........
.............................................
SW837 1.24 13.39 0.46 Synergy Additive Synergy
SW948 32.97 2.82 0.14 Synergy Synergy Synergy
T84 0.78 -3.52 -0.12 Synergy Synergy Synergy
WiDr 6.34 -0.02 0.00 Modest Additive Modest
Synergy Synergy
Tables 2-7 Key:
Cell Line = Tumor-derived cell line
gIC5o = Concentration of compound (nM) required to cause 50% growth inhibition
Ymin = The minimum cellular growth in the presence of Compound B (relative to
DMSO control) as
measured by % of that at T=O (number of cells at time of Compound B addition).
A negative number
indicates a net loss of cells relative to that at T=O.
Ymin /To = Ymin value divided by the TO value whereas the Ymin is derived from
the concentration-response
curve and the TO value represents the number of cells at the time of compound
addition (CTG
measurement).
EOHSA= Excess over highest single agent determination
BLISS = Bliss synergy determination
Comb Index = Combination Index score
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Table 3.. Basic measures and Synergy calls for each of the Lung cell lines.
Cell IC50(nM) Ymin Ymin/T EOHSA BLISS Comb
Line g 0 Index
NCIH2122 4.01 1.34 0.08 Antagonism Antagonism Synergy
......... ...........................-----
......................................................... ---------------------
-------- -------------------------------
A549 3.00 0.32 0.06 Synergy Modest Synergy
Synergy
CaIu6 2.79 3.51 0.22 Synergy Synergy Synergy
NCIH1299 1.55 11.11 1.14 Synergy Modest Synergy
Synergy
NCIH1563 0.41 1.56 0.04 Synergy Synergy Synergy
NCIH1792 2.89 1.34 0.05 Synergy Modest Synergy
.. ______ Synergy .
NCIH2O30 0.92 8.19 0.43 Synergy Modest Synergy
Synergy
NCIH2170 3.25 4.05 0.22 Synergy Modest Synergy
Synergy------..
NCIH2228 2.95 3.29 0.28 Synergy Synergy Synergy
NCIH23 1.11 7.72 0.47 Synergy Synergy Synergy
NCIH358 4.97 2.40 0.11 Synergy Synergy Synergy
NCIH460 1.91 5.11 1.25 Synergy Modest Synergy
Synergy
NCIH520 5.97 15.31 1.13 Synergy Synergy Synergy
SKMESI 2.91 0.09 0.00 Synergy Synergy Synergy
Table 4. Basic measures and Synergy calls for each of the Pancreatic cell
lines.
Cell glC50(nr) Ymin Ymin/T EOHSA BLISS Comb
Line 0 Index
Modest
BxPC3 0.43 0.81 0.02 Synergy Synergy ..Synergy
Modest
__Capan1.... .................... 1.17 ........ 12.34 0.37 Synergy
.........Synergy Synergy ...... HUPT4 0.22 3.77 0.12 Synergy Synergy Synergy
Modest
MiaPaCa 5.10 8.10 1.03 Synergy Synergy ---Synergy
SW1990 5.28 0.21 .......Synergy ...... ygy Modest
YAPC 7.34 .3.28
6.35 0.81 Synergy Synergy Synergy
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Study #2. Breast Cancer Cell Lines Analyzed for Estrogen Receptor
Experimental Preparation(s)
Combination drug tests with the MEK inhibitor (Compound A) and the P13K
inhibitor (Compound B) were conducted using a panel of cell lines from human
breast
cancers (n = 10)(Table 1). Cell lines were purchased commercially [from ATCC
(Manassas, VA, USA) or DSMZ (Braunschweig, Germany)] and grown in RPMI-1640
supplemented with 2 mM glutamine, 1 mM sodium pyruvate and 10% fetal bovine
serum
and maintained at 37 C and 5% CO2 in a humid incubator.
Experimental Protocol(s)
Fixed Ratio Drug Combination Assay
The dilution design of the Fixed Ratio Drug Combination Assay can be seen in
Figure 1. First, the test compounds were prepared as 10 mM stocks in 100%
dimethyl
sulfoxide (DMSO). Further dilutions of the compounds were made with DMSO. The
first
test compound (designated as Compound 1) is diluted horizontally in a 96 well
microtiter
plate in rows B-E using a 3-fold dilution series for 10 dilution points. A
second test
compound (designated as Compound 2) is diluted horizontally in a separate 96
well
microtiter plate in rows D-G using a 3-fold dilution series for 10 dilution
points. The two
compounds are combined using equal volumes from each drug plate into cell
culture
media. This results in a 1:50 dilution of the drugs in the cell culture media.
Compound 1
is individually titrated in rows B and C, while only Compound 2 is dosed in
rows F and G
of the plate. An additional 1:10 dilution of the drugs is performed in cell
culture media
prior to addition to the cells. Drug addition to the cells results in a
further 1:2 dilution of
drugs. The total dilution of the drug plate to the cells is 1:1000. The final
dosing
concentration range for GSK2126458A was 0.008 - 150.0 nM and was 0.013 - 250.0
nM
for GSK1120212B. The positive control consists of culture media with DMSO at
0.1 %
and cells and no drug. The negative control consists of culture media with
DMSO at
0.1%. solution.
Assays were performed in 96 well microtiter plates with appropriate seeding
densities estimated from previous studies of each cell line. Following dosing,
the cell
lines are incubated at 37 C, 5% CO2 in humid air for 72 hours. Cell
proliferation was
measured using the CellTiter Glo (Promega Corporation, Madison, WI, USA)
reagent
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according to the manufacturer's protocol. The plates are treated with
CellTiter Glo
solution and are analyzed for RLU (relative light units) using a Molecular
Devices
SpectraMax M5 (Sunnyvale, CA, USA) plate reader.
Data Analysis
The percent intensity values were used in model 205 of XLfit in Microsoft
Excel to
using a 4 parameter logistical fit to calculate response metrics, including
the midpoint of
the growth window g1C50, number of cells at time zero (To), and the intensity
value at the
bottom of the response curve Ymin Each combination experiment contains a
replicate for
the two compounds as single agents as well as a technical replicate for the
combination.
Average values were used for subsequent analysis.
Three independent metrics were used to analyze the combinatorial effects on
growth inhibition of Compound A and Compound B. These include i.) Excess over
Highest Single Agent (EOHSA; Borisy et al, 2003; FDA 21 CFR 300.50), ii.)
Bliss synergy
and iii.) Combination Index (CI). Descriptions of these three metrics and
methods for their
calculation are described above. Also, criteria used to determine the degree
of synergy
by each metric is also found above. For EOHSA and Bliss, a synergy score must
be
seen in both technical replications within an experiment to make an
appropriate
designation (synergy, modest synergy, etc). Briefly, a cell line was
considered
synergistic when at least one of the three metrics (Cl, Bliss Synergy, EOHSA)
scored in
the synergistic range as stated above.
Estrogen Receptor (ER) and Progesterone receptor (PR) transcript abundance
was measured for all cell lines using the Affymetrix U133 Plus2 GeneChips in
triplicate.
Transcript abundance was estimated by normalizing all probe signal intensities
were
normalized to a value of 150 using the mass algorithm in the Affymetrix
Microarray
Analysis Suite 5Ø For subsequent analysis, a representative probe was chosen
and the
average probe intensity was used for triplicates.
Results
A comprehensive categorization of the degree of synergy was done for each cell
line treated with the combination Compounds A and B.
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Table 5. Scores Panel of breast cancer cell lines used in combination studies.
Cell Line Organ Site Diagnosis/Histology
DU4475 Breast Carcinoma
EFM19 Breast Carcinoma
HCC1954 Breast Carcinoma
HCC70 Breast Carcinoma
MT3 Breast Carcinoma
MX1 Breast Carcinoma
NCI-ADR-RES Breast Carcinoma
UACC893 Breast Carcinoma
T47D Breast Carcinoma
ZR-75-1 Breast Carcinoma
Table 6. Basic measures and Synergy calls for each of the breast cancer cell
lines.
Cell Line gIC50(nM) Ymin Ymin/To EOHSA BLISS Comb
Index
DU4475 0.12 -0.29 -0.02 No Synergy No Synergy Modest
Synergy
EFM19 3.43 14.97 0.49 No Synergy No Synergy N/A
HCC1954 6.53 0.32 0.03 Synergy Synergy Synergy
HCC70 0.31 -0.63 -0.02 Synergy Synergy Synergy
.__
.......................................................
......................................................
...........................................................
MT3 4.47 5.59 0.39 Synergy Synergy Synergy
MX1 5.99 13.99 1.04 No Synergy No Synergy N/A
NCI-ADR- 49.01 36.28 2.17 Synergy Synergy N/A
RES
... .......................................... .... ......... ......... -------
- ............................. .__.. .... .........
....................................................
UACC893 3.44 -0.32 -0.01 Modest Modest N/A
Synergy Synergy
T47D 0.93 23.68 0.97 Synergy Synergy N/A
ZR-75-1 0.03 2.73 0.06 No Synergy No Synergy Synergy
Table 7. Panel of breast cell lines (n = 10), ER/PR transcript abundance
measurements used in
combination experiments for Compound B and Compound A.
Estrogen Receptor Progesterone
CL Name Expression (mass) Receptor
Expression mass
HCC70 75 33
DU4475 32 30
HCC1954 128 34
NCI-ADR-RES 42 120
UACC893 114 37
EFM19 2122 1333
MT3 34 25
MX1 1318 74
T47D 1329 3621
ZR-75-1 822 1103
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Study #3. In vitro cell growth inhibition and apoptosis induction by Compounds
A & B in a
panel hepatocellular carcinoma cell lines and a panel breast cancer cell lines
analyzed for
Her2 DNA copy number changes
Cell lines and growth conditions
Human tumor cell lines from hepatocellular carcinoma (HCC), C3A, Hep3B,
HepG2, PLC/PRF/5, SNU182, SNU387, SNU398, SNU423, SNU449 and SNU475 were
purchased from the ATCC. Human breast tumor cell lines, HCC2218, HCC1419, BT-
474,
SK-BR-3, UACC893, JIMT-1, MDA-MB-361, HCC202, MDA-MB-175-VII, HCC1569,
HCC1937, HCC38, MDA-MB-157, HCC1954, HCC1500, BT483, KPL-1, SUM225 and
ZR-75-1 from ATCC, SUM52 and SUM190 from Asterand, PLC (Detroit MI), were
cultured in RPMI 1640 medium containing 10% FBS; SKBR3-W13 and BT-474-J4
cultured in RPMI 1640 medium containing 10% FBS and 1 pM lapatinib; KPL4 line
was
kindly provided by Dr Junichi Kurebayashi (Kawasaki Medical School, Okayama,
Japan)
and cultured in DMEM containing 5% FBS. JIMT-1 from European Collection of
Cell
Cultures (UK), is a line derived from a patient clinically resistant to
trastuzumab
(Herceptin(D). SK-BR-3-W1 3 is a single cell clone isolated by a cloning
cylinder after a
single treatment of SK-BR-3 cells with 0.5 pM lapatinib. BT-474-J4 is a single
cell clone
derived from a pool of BT-474 cells that were selected to grow in lapatinib to
a
concentration of 3 pM.
Cell growth inhibition assay and combination data analysis
Cells were seeded in a 96-well tissue culture plate (NUNC 136102) of RPMI
medium containing 10% FBS at 500-2,000 cells per well. Approximately 24 hours
after
plating, cells were exposed to ten, two-fold or three-fold serial dilutions of
either
Compound A or B or the combination of the two agents at a 2:1 molar ratio
(Compounds
A and B respectively). In some cases, cells were grown in RPMI media
containing 10%
FBS and in the presence or absence of 2 ng/mL hepatocyte growth factor (HGF).
Cells
were incubated in the presence of compounds for 3 days. ATP levels were
determined
by adding Cell Titer Glo (Promega) according to the manufacturer's protocol.
Briefly,
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Cell Titer Glo was added to each plate, incubated for 20 minutes then
luminescent
signal was read on the SpectraMax L plate reader with a 0.5 sec integration
time. All
assays were run at least in duplicate.
Inhibition of cell growth was estimated after treatment with compound or
combination of compounds for three days and comparing the signal to cells
treated with
vehicle (DMSO). Cell growth was calculated relative to vehicle (DMSO) treated
control
wells. Concentration of compound that inhibits 50% of control cell growth
(IC50) was back-
interpolated when y = 50% of DMSO treated control wells using nonlinear
regression with
the equation:
A+(B-A)
Y= +(C)D
X
where A is the minimum response (ymin), B is the maximum response (ymax), C is
the
inflection point of the curve (EC50) and D is the Hill coefficient.
Combination effects on potency were evaluated using the Combination Index (CI)
and Excess Over Highest Single Agent (EHOSA) methods.
In this study, co-administration of Compounds A & B exhibit a synergistic
interaction in a specific cell line to potency or on the response scale, if
the Cl <0.9 or the
EOHSATD >0.
Cell apoptosis assays- caspase-3/7 activation and DNA fragmentation
For investigation of the induction of apoptosis, all cell lines were plated at
5,000
cells per well in a 96-well tissue culture plate and allowed to attach for
approximately 24
hours. Cells were then treated with compounds as described above. 24 hours
after
compound treatment, the levels of active caspase 3 and caspase 7 were
determined with
the Caspase GIoTM 3/7 (Promega, cat G3093) according to the instructions
provided by
the manufacturer. 48 hours after treatment with compound, levels of apoptosis
were
estimated using the Roche Cell Death ELISA (Roche, Inc., Basel, Switzerland;
Cat. No.
11 774 425 001) following the instructions provided by the manufacturer.
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For the purposes of molecular characterization of selected cell lines, the
expression levels of several key proteins were measured by western blot. These
included E-cadherin (CDH-1), vimentin (VIM), HER3 STAT3, MET, AKT and ERK1/2.
Actin was used as a control in each case
DNA COPY NUMBER
DNA Copy number data on the HER2 gene was collected for all breast cancer cell
lines
using the Affymetrix 500K chip (Affymetrix Inc, Sunnyvale, CA). Briefly DNA
was
extracted from each line, digested with the restriction enzyme Nsp or Sty,
ligated to an
adaptor and amplified by PCR. After PCR, DNA was fragmented, labeled,
denatured,
and hybridized to the Affymetrix 500K chip. Upon completion of hybridization,
each assay
was washed and stained. Image data were acquired. Similarly collected data
from a
panel 10 diploid non-tumorigenic lymphoblastic cell lines were used to
calculate DNA
copy number. All 'SNP Chip' images ('CEL files'), were extracted, read and
normalized
using the dChip software package (Lin et al. 2004. Bioinformatics. 20:1233-
40). SNP-
wise `copy-number ratios' (loge scale) were calculated for all cancer cell
lines using the
lymphoblastic reference panel and analyzed by circular binary segmentation to
reduce
noise (Olshen et al. 2004. Biostatistics. 5:557-72). Cell lines with loge
ratios of HER2 >
0.65 were considered HER2+.
Results
Effects of cell growth inhibition and apoptosis on hepatocellular carcinoma
cell lines by
Compound A and Compound B combination
The genetic backgrounds and protein expression analyzed by Western blot in 10
hepatocellular carcinoma (HCC) cell lines were shown in Figure 2. The cell
lines C3A,
Hep3B, HepG2, PLC/PRF/5 and SNU182 express high levels of CDH-1 and extremely
low to low levels of VIM, whereas SNU387, SNU398, SNU423, SNU449 and SNU475
cell
lines express relatively high levels of VIM and extremely low levels of CDH-1.
High levels
of CDH-1 and low or no VIM is characteristic of epithelial cells, while high
VIM and low
CDH-1 is characteristic of mesenchymal cells. Therefore, C3A, Hep3B, HepG2,
PLC/PRF/5 and SNU182 are defined as epithelial-like and SNU387, SNU398,
SNU423,
SNU449 and SNU475 as mesenchymal-like cells. This is consistent with the fact
that
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HER3 is highly expressed in the epithelial-like HCC lines and AXL is highly
expressed in
mesenchymal-like cells (data also shown in Figure 2). STAT3, AKT and ERK1/2
(total
protein) were expressed at a similar level in epithelial-like and mesenchymal-
like cell
lines, while MET expression was variable, but not differentially-associated
with either
group of cells. Phosphorylation/activation of AKT is preferentially observed
in
mesenchymal-like cell lines, with higher levels of pAKT-S473 than pAKT-T308.
pERK1/2
was also differentially, but not exclusively, present in mesenchymal-like
cells.
The effects of cell growth inhibition by Compound A, Compound B and their
combination
were determined in 10 HCC cell lines. The mean IC50s (from at least two
independent
experiments) and the combination effects at IC50s are summarized in Table 8.
Three
epithelial-like HCC cell lines (HepG2, C3A and Hep3B) were strongly sensitive
to cell
growth inhibition by Compound A (IC50<37 nM), and SNU 182 and PLC/PRF/5
epithelial-
like cell lines were weakly sensitive to Compound A (IC50=1.2 -2.8 pM). Two
mesenchymal-like HCC cell lines (SNU387 and SNU423) were moderately sensitive
to
cell growth inhibition by Compound A (IC50=74-577 nM) while three mesenchymal-
like cell
lines (SNU398, SNU449 and SNU475) were not sensitive to cell growth inhibition
by
Compound A. All 10 HCC lines were sensitive to cell growth inhibition by
Compound B
(IC50 <103 nM). Furthermore, combination treatment with Compound A and
Compound B
(1:2 ratio) showed strong synergy as demonstrated by the combination index
values
ranging from 0.22 to 0.78 or greater than the best single agent by EOHSATD
analysis (5--
20 ppt) and EOHSA analysis (12-27 ppt) in 8 of 10 HCC cell lines. The presence
of HGF
had no consistent effect on responsiveness to either drug alone or in
combination.
These 10 HCC lines were further evaluated for the ability of Compound A,
Compound B
or the combination of Compound A and Compound B to induce apoptosis as
determined
by caspase 3/7 activities. Activation of caspase 3 is a hallmark of induction
of apoptosis.
Representative caspase 3/7 activity curves for these cells are provided in
Figure 3. All cell
lines except SNU182 showed strong enhancement of apoptosis by combination
treatment
with Compound A and Compound B relative to single agent treatment with
Compound A
or Compound B. SNU182 cells showed moderate enhancement of apoptosis by
combination treatment with Compound A and Compound B relative to their single
agent
treatment.
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Effects of cell growth inhibition on human breast tumor cell lines measured
for Her2 levels
by Compound A and Compound B combination
Analysis of copy number alterations in the HER2 gene identified 14 as HER2
positive
(HER2+) breast tumor lines. These were BT474, BT474-J4, HCC1419, HCC1954,
HCC202, HCC2218, JIMT-1, KPL-4, MDA-MB-361, SK-BR-3, SK-BR-3-W13, SUM190,
SUM225 and UACC893. A total of 10 were considered HER2 negative (HER2-). These
include BT483, HCC1500, HCC1569, HCC1937, HCC38, KPL-1, MDA-MB-157, MDA-
MB-175-VII, ZR-75-1 and SUM52.
The effects of cell growth inhibition by Compound A, Compound B and their
combination
were determined these 25 cell lines, The mean IC50 values (from at least two
independent
experiments) and the combination effects at IC50 values are summarized in
Table 9.
Cell lines SUM52 and MDA-MB-17511 are sensitive to Compound A with IC50 values
of
less than or equal to 0.099 pM. In contrast, all lines except HCC1937, SK-Br-3-
W13 and
MDA-MB-157 are sensitive to Compound B with IC50 < 0.1 pM. The combination of
Compound A and Compound B showed synergy with combination index (CI) values
between 0.48 and 0.83 and greater than the most active single agent analysis
(EOSHA)
between 15 and 25 ppts in SUM52, HCC1954 (HER2+) and MDA-MB-17511 (HER2-) cell
lines. The combination of Compound A and Compound B also showed a benefit of
greater than the most active single agent analysis (EOSHA) between 10 and 15
ppts in a
subset of HER2+ (SUM190, HCC202) and HER2- lines (MDA-MB-157, HCC1937). The
combination of Compound A and Compound B showed a comparable effect to the
most
active single agent in the rest of the lines. The mean EOHSA score for Her2+
lines (n =
14) was 9.1 ( 7.4), while the mean score for the Her2- line s (n = 10) was
6.9 ( 7.2).
These EOHSA scores did not significantly differ between groups (p = 0.45; t-
test).
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Table 8. Cell growth inhibition by Compound A, Compound B and their
combination in
human hepatocellular carcinoma tumor cell lines.
HGF Single agent (IC50, M) Combination (IC50, NM) Combination Effect (A:
B=1:2)
Cell line
(2ng/ml) EOHSATD, EOHSA,
Compound A Compound B Compound A Compound B Cl ppt ppt
- 0.001 0 .000 0.009 0.008 0.001 0 .000 0.001 0.001 0.72+0.05 <0 13.5+5.0
HepG2
+ 0.003 0.001 0.009 0.005 0.001 0.000 0.002 0.001 0.57+0.12 8.2+1.1
22.7+7.3
- 0.010 0.003 0.013 0.001 0.001 0.000 0.002 0 .000 0.30 0.06 11.4 3.6
20.6 1.4
C3A
+ 0.036 0.011 0.013 0.001 0.002 0.000 0.004 0.001 0.39 0.03 11.1 2.2
16.5 2.1
' i - 0.026 0.008 0.028 0.002 0.005 0.003 0.010 0.005 0.61 0.26 12.6 9.9
19.2 9.9
Hep3B
+ 0.037 0.008 0.057 0.020 0.006 0 .000 0.012 0.001 0.44+o.18 19.3+10.2
25.0+9.7
W SNU182 1.758 0.224 0.017 0.001 0.003 0.000 0.005 0.000 0.31 0.01
13.4+2.o 20.5+2.7
+ 1.282 1.223 0.017 0.004 0.002 o.ooo 0.005 0.000 0.29 0.09 14.9 6.0
21.5 6.2
- 1.604 0.509 0.018 0.012 0.002 0.001 0.005 0.002 0.32+0.12 15.4 10.6
22.2+1o.8
PLC/RF/5
+ 2.871 1.556 0.015 0.001 0.003 0.001 0.006 0.001 0.41 0.06 8.4 1.8 15.3
1.4
- 0.218 0.130 0.023 0.016 0.006 0.003 0.011 0.007 0.55 0.05 4.8 1.8 11.7
2.1
SNU423 + 0.577 0.569 0.021 0.007 0.008 0.004 0.016 0.008 0.78 0.09 <0
5.9+2.7
Y >5 0.024 0.005 0.013 0.007 0.026 0.013 NA <0 -0.6 5.5
SNU449
m + >5 0.024 0.011 0.015 0.003 0.029 0.006 NA <0 -3.5 4.3
E >5 0.050 0.019 0.016 0.002 0.033 0.003 NA <0 9.0 11.4
SNU475 + >5 0.039 0.017 0.018 0.003 0.036 0.007 NA <0 0.3 16.9
U - >5 0.098 0.021 0.009 0.003 0.020 0.007 NA 20.1 4.8 26.9+5.7
SNU398
+ >5 0.083 0.002 0.008 o.oo1 0.016 0.001 NA 20.1 1.7 26.8 2.0
- 0.074 0.046 0.103 o.oio 0.006 0.002 0.012 0.004 0.22 o.oo 14.2 4.9
21.0+o.8
SNU387
+ 0.094 0.021 0.071 0.010 0.008 0.003 0.016 0.005 0.34 0.13 11.3 4.9
15.4 4.8
Table 8 Key:
HGF: Hepatocyte Growth Factor; in the presence, in the absence.
IC50: the concentration of Compound(s) that reduces cell growth by 50%;
Cl; Combination Index; NA = not applicable
EOHSATD: Excess Over Highest Single Agent at Total Dose, measured as a
percentage
EOHSA: Excess over Highest Single Agent, measured as a percentage
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Table 9. Cell growth inhibition by Compound A, Compound B and their
combination
in breast tumor cell lines.
Single agent (IC50, NM) Combination (A:B=1:1)
Breast cell HER2
lines Compound
Compound A ComBBound A or B (IC50, Cl EOHSA
S pp pt
NM). (AorB; )
0.011 0.005 0.48
HCC1 954 HER2+ 1.018 0.839 0.003 0.002 0.07 24.5 3.3
0.006 0.003
SUM190 HER2+ >1 0.003 0.001 NA 15.0 5.6
0.045 0.013
HCC202 HER2+ >1 0.004 0.000 NA 11.5 0.3
0.018 0.009
HCC2218 HER2+ >1 0.013 0.002 NA 9.6 9.4
0.028 0.018
JIMT-1 HER2+ >1 0.005 0.002 NA 9.3 6.7
0.003 0.002
UACC893 HER2+ >1 0.003 0.002 NA 8.6 3.8
0.129 0.091
SK-BR-3-W13 HER2+ >1 0.099 0.107 NA 6.8 5.2
0.014 0.012
BT474-J4 HER2+ >1 0.008 0.011 NA 5.6 4.9
0.009 0.007
MDA-MB-361 HER2+ >1 0.002 0.002 NA 4.3 4.8
0.024 0.020
HCC1419 HER2+ >1 0.014 0.011 NA 3.7 3.1
0.003 0.003
KPL4 HER2+ >1 0.001 0.001 NA -1.7 7.8
0.024 0.024
SK-BR-3 HER2+ >1 0.020 0.016 NA -1.6 2.4
0.013 0.011
SUM225 HER2+ >1 0.014 0.011 NA 1.2 2.7
0.030 0.033
BT474 HER2+ >1 0.007 0.010 NA -0.9 1.1
0.004 0.002 0.83
SUM52 HER2- 0.009 0.005 0.000 0.000 0.11 22.7 5.5
MDA-MB-175- 0.007 0.004 0.60
VII HER2- 0.099 0.097 0.001 0.001 0.08 15.0 5.1
0.077
MDA-MB-157 HER2- >1 >1 0.047 NA 15.5 4.2
0.114 0.069
HCC1937 HER2- >1 0.063 0.048 NA 11.0 3.8
0.008 0.007
KPL1 HER2- >1 0.004 0.002 NA 2.0 3.4
0.006 0.005
ZR-75-1 HER2- >1 0.001 0.001 NA 3.2 0.5
0.106 0.082
BT483 HER2- >1 0.081 0.083 NA 4.4 4.4
0.061 0.035
HCC1500 HER2- >1 0.036 0.012 NA 9.5 5.8
0.095 0.059
HCC38 HER2- >1 0.034 0.005 NA 9.7 7.8
0.076 0.095
HCC1 569 HER2- >1 0.042 0.082 NA -1.6 7.5
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Table 9 Key:
HER2: HER2+ = HER2 positive, log2 ratios of HER2 DNA copy number > 0.65; HER2-
= HER2
negative, log2 ratios of HER2 DNA copy number < 0.65.
*IC50: the concentration of Compound A in the presence of equal molar Compound
B that reduces cell
growth by 50%;
Cl; Combination Index; NA = not applicable; EOHSA: Excess over Highest Single
Agent, measured as
a percentage.
Example 1 - Capsule Composition
An oral dosage form for administering a combination of the present
invention is produced by filing a standard two piece hard gelatin capsule with
the
ingredients in the proportions shown in Table I, below.
Table I
INGREDIENTS AMOUNTS
N-{3-[3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8- 5mg
dimethyl -2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-
d]pyrimidin-1(2H)-yl]phenyl}acetamide dimethyl sulfoxide
(the dimethyl sulfoxide solvate of Compound A)
2,4-difluoro-N-{2-(methyl oxy)-5-[4-(4-pyridazinyl)-6- 10mg
quinolinyl]-3-pyridinyl}benzenesulfonamide (Compound B)
Mannitol 50 mg
Talc 25 mg
Magnesium Stearate 2mg
Example 2 - Capsule Composition
An oral dosage form for administering one of the compounds of the
present invention is produced by filing a standard two piece hard gelatin
capsule with
the ingredients in the proportions shown in Table II, below.
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Table II
INGREDIENTS AMOUNTS
N-{3-[3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8- 5mg
dimethyl -2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-
d]pyrimidin-1(2H)-yl]phenyl}acetamide dimethyl sulfoxide
(the dimethyl sulfoxide solvate of Compound A)
Mannitol 55 mg
Talc 16 mg
Magnesium Stearate 4 mg
Example 3 - Capsule Composition
An oral dosage form for administering one of the compounds of the
present invention is produced by filing a standard two piece hard gelatin
capsule with
the ingredients in the proportions shown in Table III, below.
Table III
INGREDIENTS AMOUNTS
2,4-difluoro-N-{2-(methyl oxy)-5-[4-(4-pyridazinyl)-6- 10mg
quinolinyl]-3-pyridinyl}benzenesulfonamide (Compound B)
Mannitol 50mg
Talc 25mg
Magnesium Stearate 2mg
Example 4 - Tablet Composition
The sucrose, microcrystalline cellulose and the compounds of the
invented combination, as shown in Table IV below, are mixed and granulated in
the
proportions shown with a 10% gelatin solution. The wet granules are screened,
dried, mixed with the starch, talc and stearic acid, then screened and
compressed
into a tablet.
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Table IV
INGREDIENTS AMOUNTS
N-{3-[3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8- 5mg
dimethyl -2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-
d]pyrimidin-1(2H)-yl]phenyl}acetamide dimethyl sulfoxide
(the dimethyl sulfoxide solvate of Compound A)
2,4-difluoro-N-{2-(methyl oxy)-5-[4-(4-pyridazinyl)-6- 10mg
quinolinyl]-3-pyridinyl}benzenesulfonamide (Compound B)
Microcrystalline cellulose 60mg
sucrose 5mg
starch 10mg
talc 5mg
stearic acid 2mg
Example 5 - Tablet Composition
The sucrose, microcrystalline cellulose and one of the compounds of
the invented combination, as shown in Table V below, are mixed and granulated
in
the proportions shown with a 10% gelatin solution. The wet granules are
screened,
dried, mixed with the starch, talc and stearic acid, then screened and
compressed
into a tablet.
Table V
INGREDIENTS AMOUNTS
N-{3-[3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8- 5mg
dimethyl -2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-
d]pyrimidin-1(2H)-yl]phenyl}acetamide dimethyl sulfoxide
(the dimethyl sulfoxide solvate of Compound A)
Microcrystalline cellulose 30mg
sucrose 4mg
starch 2mg
talc 1 mg
stearic acid 0.5mg
Example 6 - Tablet Composition
The sucrose, microcrystalline cellulose and one of the compounds of
the invented combination, as shown in Table VI below, are mixed and granulated
in
the proportions shown with a 10% gelatin solution. The wet granules are
screened,
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dried, mixed with the starch, talc and stearic acid, then screened and
compressed
into a tablet.
Table VI
INGREDIENTS AMOUNTS
2,4-difluoro-N-{2-(methyl oxy)-5-[4-(4-pyridazinyl)-6- 10mg
quinolinyl]-3-pyridinyl}benzenesulfonamide (Compound B)
Microcrystalline cellulose 60mg
sucrose 5mg
starch 10mg
talc 5mg
stearic acid 2mg
While the preferred embodiments of the invention are illustrated by the
above, it is to be understood that the invention is not limited to the precise
instructions herein disclosed and that the right to all modifications coming
within the
scope of the following claims is reserved.
